Secretion of proteins by eukaryotic cells into the culture medium involves transfer of the proteins through the various membrane-enclosed compartments constituting the secretory pathway. First, the proteins are translocated into the lumen of the endoplasmic reticulum (ER). From there on, the proteins are transported in membrane vesicles to the Golgi complex and from the Golgi complex to the plasma membrane. The secretory pathway involves several steps in which vesicles containing the secreted proteins are pinched off from the donor membrane, targeted to and fused with the acceptor membrane. At each of these steps, the function of several proteins such as chaperones or folding enzymes is needed in order to perform adequate maturation of the proteins including glycosylation and disulphide bridge formation. The extracellular proteins mature in the oxidizing environment of the ER where they become core glycosylated and this glycosylation process is subsequently completed in the Golgi complex.
Several attempts have been made to increase protein secretion in eukaryotes. A common approach to increase secretion of heterologous proteins is to use signal sequences (see for example EP 0215 594). The conventional secretory pathway in eukaryotic cells, as outlined above, is by definition adapted to the maturation of extracellular proteins. The maturation of intracellular proteins is realized in the reducing environment of the cytoplasm having specific chaperones and folding proteins. Production of proteins, especially intracellular proteins, in an industrial setting is still a difficult task due to the low yield of proteins caused by the inefficiency of both the secretion and the down stream processing (Hopkins T R. Physical and chemical cell disruption for the recovery of intracellular proteins. Bioprocess Technol. 1991; 12:57-83.)
Due to the growing industrial importance to produce proteins and to the poor efficiency of both secretion and down stream processing pathways, there is still a need to obtain improved process for production of proteins in eukaryotic cells. The present invention provides a novel method to produce proteins with high efficiency.